The release of neurotransmitters is a fundamental step in neuronal communication and is probably an important site of plasticity during development or learning. Neurotransmitters are released, from storage organelles called synaptic vesicles, via the process of calcium-regulated exocytosis. The availability of synaptic vesicles for exocytosis is tightly regulated and seems to be determined in part by "priming" processes that occur after a vesicle docks at the presynaptic membrane and before its Ca-dependent fusion with it. The general goal of this research is to define the molecular mechanisms underlying the exocytosis of synaptic vesicles focusing in particular on the process of vesicle priming. The experiments proposed here will test, in autaptically connected hippocampal neurons in culture, the hypothesis that an ATP- dependent priming process is involved in neurotransmitter release. The time course of transmitter release will be monitored following an instantaneous rise in the Ca concentration due to photolysis of a "caged" Ca compound. The hypothesis predicts that a multiphasic release time course will be evoked, arising from the sequential exocytosis of primed vesicles followed by unprimed ones, and that inhibition of ATP hydrolysis will inhibit only the release of unprimed vesicles. This kinetic assay will also be used to determine whether two cytosolic proteins critical in production of phosphoinositides play a role in priming. These experiments are of general interest because they will help identify the molecular basis for a process that is central to the ability of the brain to process information.